α-glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglucosylase i :...

Eur. J. Biochem. 157,539-545 (1986) 0 FEBS 1986

a-Glucan synthesis on a protein primer, uridine diphosphoglucose : protein transglucosylase I Separation from starch synthetase and phosphorylase and a study of its properties

Silvia MORENO, Carlos E. CARDINI and Juana S. TANDECARZ Instituto de Investigaciones Bioquimicas ‘Fundacion Campomar’ and Facultad de Ciencias Exactas y Naturales, Buenos Aires

(Received January 3/March 21,1986) - EJB 86 0005

It was found that the DEAE-cellulose-treated UDP-Glc : protein transglucosylase I catalyzing the first step (reaction 1) in the formation of a-glucan bound to protein in potato tuber is not only specific for the glucosyl donor but also for the endogenous acceptor. A single radioactive 38-kDa macromolecular component appeared during denaturing polyacrylamide gel electrophoresis of reaction 1 product. The labeled component is probably the polypeptide subunit of the endogenous acceptor which is being glucosylated. The radioactivity incorporated in reaction 1 product was isolated from a protease digest as a low-molecular-mass glucopeptide fraction. A fl-elimination reaction carried out in the presence of a reducing agent demonstrated that only one glucosyl moiety is transferred from UDP-Glc to the aminoacyl residue, thus forming an O-glucosidic linkage. 3H-labeled sodium borohydride showed that serine and threonine are involved in the peptide bond to glucose. Ion-exchange chromatography on DEAE-cellulose, affinity chromatography on concanavalin-A - Sepharose, gel filtration on Sephacryl S-300 and sucrose density gradient centrifugation failed to separate the enzyme catalyzing reaction 1 from the endogenous acceptor.

Past work from our laboratory has shown the occurrence of an enzymatic system in a particulate fraction of potato tuber capable of synthesizing cr-1 ,Cglucosidic chains co- valently bound to protein in the presence of appropriate concentrations of UDP-Glc, ADP-Glc or glucose-l-P as glucosyl donors [l, 21. This protein-bound glucan synthesis takes place in the absence of added polysaccharide primer. A protein acceptor also seems to be involved in the biosynthesis of glycogen by rat liver [3] and heart [4], by bovine retina [5 ] and by Escherichia coli [6] as well as in the biosynthesis of a 8-1,3- glucan (paramylon) by Euglena gracilis [A.

Our previous results have suggested that a-glucan synthesis on a protein primer could be related to starch biosynthesis and, according to experimental evidence, it might be involved in a two-step pathway in the potato tuber [ti, 91. The first step is the transfer of glucosyl moieties specifically from

Correspondence to J. S. Tandecarz, Instituto de Investigaciones Bioquimicas, ‘Fundacion Campomar’, Antonio Machado 151, RA-1405 Buenos Aires, Argentina

Dedicated to Professor Dr Luis F. Leloir on the occassion of his 80th anniversary.

Abbreviations. ADP-Glc, adenosine 5’-diphospho-glucose; ConA, concanavalin A; glucose-l-P, glucose l-phosphate; PAGE, polyacrylamide gel electrophoresis; HI04/Schiff, periodic acid/Schiff reagent; SDS, sodium dodecyl sulfate; CIJAcOH, trichloroacetic acid ; TLC, thin-layer chromatography.

Enzymes (IUB Recommendations 1984). UDPglucose : protein 4-~1-~-glucosyltransferase or UDP-Glc : protein transglucosylase I (EC 2.4.1.112); alkalinephosphatase (EC 3.1.3.1); cc-amylase (EC 3.2.1.1); j?-amylase (EC 3.2.1.2); catdase (EC 1.1 1.1.6); j?-galactosidase (EC 3.2.1.23); lactate dehydrogenase (EC 1.1.1.27); phosphorylase or GI-1 ,4-glucan: orthophosphate glucosyltransferase (EC 2.4.1 .I); starch synthetase or ADPglucose : starch a-4-glucosyltransferase (EC 2.4.1.21); subtilisin (EC 3.4.21.14).

UDP-Glc to an endogenous protein, thus allowing the formation of a peptidyl-glucose linkage. This enzyme is referred to as UDP-Glc: protein transglucosylase I and its activity can be observed at micromolar concentrations of the sugar nucleo- tide owing to its high affinity for the glucosyl donor. In the second step, the glucosylated protein already formed serves as a ‘primer’, like glycogen or starch, for either starch synthetase or phosphorylase, naturally occurring in the potato preparation. a-l,6Glucosidic chains linked to protein are thus synthesized with millimolar concentrations of ADP-Glc, UDP-Glc or glucose-l-P [S, 91. The reactions under study can be schematically expressed as follows :

UDP-Glc + acceptor protein

Acceptor protein-Glc + n UDP(ADP)-Glc or n glucose-l-P + acceptor protein-Glc-(Glc), + n UDP(ADP) or n Pi, (2)

Although a fair proportion of the reactions catalyzed by glucosyltransferases have been studied in considerable detail, knowledge regarding the glucosyltransferases involved in the biosynthesis of protein-bound a-1 ,Cglucan is mostly limited to properties that have often been determined with crude enzyme preparations [l -91. The property of being membrane-linked, which is shown by the enzymes catalyzing both reactions involved in protein-bound glucan synthesis [l, 21, complicated the separation of the different enzymes or acceptor proteins from other membrane-bound proteins. To understand the individual biosynthetic steps that ultimately lead to formation of protein-bound glucan in more detail, it was necessary to solubilize the glucosyltransferases involved in this pathway in order to: (a) determine the specificity of the primary acceptor; (b) identify the glucopeptide linkage in the

+ acceptor protein-Glc + UDP . (1)

540

intermediate glucoprotein (reaction 1 product); (c) characterize the glucosyltransferases.

All the protein components of the system engaged in protein-bound glucan synthesis can be solubilized on increasing the ionic strength of the medium [lo]. These proteins appear to be loosely bound to the membrane surface based upon the ease with whch the enzymes can be stripped from the membrane without the intervention of detergents. In order to characterize the enzyme catalyzing reaction 1, we have isolated the UDP-Glc : protein transglucosylase I activity found in the potato tuber solubilized preparation. Its properties, as well as those of its glucosylated product (reaction 1 product), are reported here. Evidence is presented indicating that the formation of a Ser/Thr-Glc linkage is the first glucosylating event.

MATERIALS AND METHODS

Materials

UDP-['4C]Glc (268 Ci/mol) and ADP-['4C]Glc (262 Ci/ mol) were obtained as previously described [ll]. NaB3H4 (884 Ci/mol) and ['4C]glucose-l-P (156 Ci/mol) were from New England Nuclear. Sweet potato P-amylase, bacterial a-amylase (type 11-A), protease from Bacillus amyloliquefaciens (subtilisin), catalase from beef liver, rabbit muscle lactate dehydrogenase and alkaline phosphatase (E. coli, type III-S) were obtained from Sigma Chemical Co.

Sephadex G-15 and Sephacryl S-300 were purchased from Pharmacia Fine Chemicals, ConA-Sepharose 4B and a- D-glucose-l-P (dipotassium salt, grade I) were from Sigma and acrylamide and bisacrylamide, from Bio-Rad. All other reagents were of the highest quality available.

Preparation of the solubilized enzymes

Solubilized preparations were made from membranes of potato tuber (10- 15 mg/ml protein) obtained as in [l] by keeping them in 100 mM Tris/HCl buffer, pH 7.4, 5 mM 2-mercaptoethanol (buffer A) at 4°C for 48-72 h, as previously described [lo]. The mixture was centrifuged for 2 h at 140000 x g and the supernatant, containing solubilized enzymatic activities, was used immediately.

Ion-exchange chromatography using DEAE-cellulose

Following solubilization of potato tuber membranes, the supernatant (6ml) was loaded onto a column (15 cm long x 1.8 cm diameter) of DEAE-cellulose (Whatman DE 52), equilibrated with buffer A. Following loading of the sample, the column was washed with 60 ml of the equilibrating buffer. UDP-Glc : protein transglucosylase I was eluted from the resin with a gradient of KCl (0- 500 mM) in buffer A. Fractions ( 3 ml) were collected at a flow rate of0.5 ml . min-'. Aliquots were measured for enzymatic activity and the fractions showing UDP-Glc : protein transglucosylase I activity were combined and concentrated by ultrafiltration to 2 ml (peak I) over a PM-10 membrane (Amicon Inc.), stored at 0 "C and used for subsequent characterization studies.

tion of reaction 1 product) was measured as incorporation of [14C]glucose into 10% C1,AcOH-insoluble material [l , 21. Unless otherwise specified, the standard incubation mixture contains, in 50 pl total volume, 0.175 nmol UDP-[14C]Glc (75000 cpm), 6 mM MnC12 and 5-20 pg of peak I. In- cubations were carried out at 30°C for 30 min.

Primed phosphorylase activity. This was determined by adding 100 pl of the DEAE-cellulose column effluent to a medium containing 0.23 nmol ['4C]glucose-l-P (45000 cpm) and 0.4 mg oyster glycogen (Sigma) in final volume of 150 pl. After incubation for 150min at 3 0 T , 1 ml methanol was added and ['4C]glucose incorporation in the methanol precipitate was measured as already described [12].

Unprimed phosphorylase activity. This was determined by adding 50 p1 of the DEAE-cellulose column effluent to a medium containing 1 pmol ['4C]glucose-l-P (45000 cpm), 0.25 mg bovine serum albumin (Sigma) and 10 pmol Mes buffer, pH 5.65, in a final volume of 100 pl. After incubation for 30 min at 30"C, 1 ml of 10% C13AcOH was added and the [ ''C]glucose incorporation in the C1,AcOH precipitate was measured as previously reported [13].

ADP( UDP) -Glc: starch glucosyltransferase (starch synthetase) . The activity was measured by adding 150 pl of the DEAE-cellulose column effluent to a medium containing 0.18 nmol ADP-['4C]Glc (87000 cpm) or 0.24 nmol of UDP- [14C]Glc (80000 cpm) and 0.4 mg oyster glycogen in a final volume of 200 pl. After incubation for 180 min at 30"C, 1 ml methanol was added and [ 14C]glucose incorporation in the methanol precipitate was measured as already described [l].

Binding to ConA Peak I (1 ml) was loaded onto a ConA-Sepharose 4B

column (0.6 x 10 cm) in 0.1 M NaC1, 1 mM each of MgC12, MnCl, and CaC12 in buffer A (buffer B). Buffer B containing 0.2 M methyl a-glucoside was used as elution buffer for the ConA-Sepharose 4B column.

Determination of Stokes radius and sedimentation coefficients

The Stokes radius and sedimentation coefficients were determined by analytical gel filtration and sucrose density gradient sedimentation. For determination of Stokes radius, peak I (500-800 pg) was applied to a column of Sephacryl S-300 (1.1 x 116 cm), equilibrated in buffer A. The column was developed at 8 ml h-' and fractions of 1.5 ml were collected. The protein standards [cytochrome c (Stokes radius: 1.87 nm); alkaline phosphatase (2.93 nm); catalase (5.21 nm) and P-galactosidase (6.84 nm)] were run separately on the column under the same conditions. For determination of sedimentation coefficients, peak I (200 pg) or the incubation mixture for reaction 1 product formation after incubation for 30 min (200 pg) in 200 pl were applied to 4.0-ml 5 - 20% (w/v) sucrose gradients in buffer A. Standards (0.01 - 0.4 mg of each in 200 pl) were applied to an opposing gradient and centrifugation was performed for 16 h at 40000 rpm in a Beckman SW 60 rotor. Gradients were fractioned from the bottom and 10-drop fractions were collected.

Enzyme assays

UDP-Glc :protein transglucosylase I . Transfer of glucose from UDP-['4C]Glc to endogenous acceptor protein (forma-

Preparation of radioactive reaction 1 product

Reaction 1 product was obtained upon incubation of peak I protein (200 pg) with 4 pM UDP-[14C]Glc and 6 mM

541

Fraction number Fig. 1 . Separation of UDP-Glc:protein transglucosylase Ifrom the soluhilizedpreparation ojpotato tuber. 50 mg of solubilized preparation was applied to a column (15 x 1.8 cm) of DEAE-cellulose equilibrated with buffer A. After the column was washed with the same buffer, the adsorbed proteins were eluted with a linear gradient of KCl. (A) UDP-G1c:protein transglucosylase I (0), ADP-Glc starch synthetase ( A ) and UDP-Glc starch synthetase (A) activities were measured with 100 pl, 150 11 and 150 p1 of sample, respectively, taken from the fractions, as described under Materials and Methods. (B) Primed (0) and unprimed phosphorylase ( W ) were measured with 100 pl or 50 pl of sample, respectively, taken from the fractions. ( 0 ) Protein. Fractions indicated by horizontal bars were pooled

MnClz at 30°C for 30min. The incubation mixture was stopped by the addition of 10% C1,AcOH. The precipitate was collected by centrifugation and washed several times with 10% C13AcOH. After removing the remaining acid with ether, the radioactive product was solubilized in distilled water.

Other procedures

Conditions for proteolytic and amylolytic treatments were as in (1, 21. Alkaline borohydride reduction was done as in

Urea/SDS-PAGE was run according to Ziegler et al. [14]. Protein standards were identified by staining with 0.25% Coomassie blue and radiolabeled proteins by fluorography

Paper electrophoresis and descending paper chromatography were carried out on Whatman no.1 paper. One- and two-dimensional TLC were done on silica gel plates (5.0 x 20 x 0.025 cm or 20 x 20 x 0.025 cm, respectively, DC- Fertigplatten Kieselgel 60 Merck). Reducing substances were located with the alkaline-silver reagent [16], peptide material with the fluorescamine stain [17] and radioactivity in the paper strips and in the silica gel plates was scanned on a Packard radiochromatogram scanner, model 7201.

Proteins were measured by the procedure of Lowry et al. [181.

t81.

1151.

RESULTS AND DISCUSSION

Separation of UDP-Glc :protein transglucosylase I

When the solubilized preparation of potato tuber was applied to a DEAE-cellulose column and eluted with a linear gradient of KCl (0 - 0.5 M), UDP-Glc: protein transglucosylase I activity was eluted as shown in Fig. 1. Fractions 68 - 76 were combined into a pool designated peak I and concentrated by ultrafiltration. ADP-Glc and UDP-Glc starch synthetase activities eluted from the column at a higher ionic strength in a rather broad zone (Fig. 1 A). On the other hand, primed phosphorylase activity eluted as two overlapping peaks, one at 0.3 M KC1, the other slightly behind the first (Fig. 1 B). Fractions 90- 100 and 108 - 120, containing phosphorylase activity were combined into two pools, named peak IIa and peak IIb, respectively. Peak IIa contained primed as well as unprimed phosphorylase activity [13] and peak IIb contained only primed phosphorylase. Furthermore, ADP-Glc and UDP-Glc starch synthetase-primed activities are found in both peaks, as they eluted in the same range of salt concentrations (Fig. 1 A). These data indicate that DEAE- cellulose chromatography succeeded in separating the enzyme that catalyzes reaction 1 from the other glucosyl transferases present in the solubilized preparation (Fig. 1). The former enzyme was purified 20-fold over the solubilized preparation which corresponds to a nearly SO-fold purification with re-

542

gt @ /-

0 10 20 30 40

TIME (min)

6 12 i a PROTEIN ( pg 1

Fig.2. ( A ) Time course of reaction 1 product synthesis in the presence of (0) 6 pg, (a) 12 pg or (0) 18 pgprotein (peak I ) . ( B ) Effect of enzyme concentration on the reaction rate. Incubations were carried out as described under Materials and Methods

spect to the particulate preparation [lo]. The availability of the partially purified activity should facilitate detailed studies on enzymatic properties of this enzyme and its role in the initiation of protein-bound a-glucan synthesis in potato tuber.

Time course

Synthesis of reaction 1 product catalyzed by peak I pro- ceeded rapidly (Fig. 2A), as already described for the enzyme in the particulate [l, 21 and solubilized [lo] preparations. At 30 "C, the incorporation of ['4C]glucose in C1,AcOH-insoluble material was completed in approximately 15 min. In- creased formation of labeled reaction 1 product was observed with increased amounts of peak I. Thus, UDP-G1c:protein transglucosylase I activity in peak I was proportional to the amount of protein added to 18 pg (Fig.2B), as opposed to the non-linear enzyme assay which is found with most mammalian glycosyltransferases [19]. The kinetics of the enzyme in peak I suggests the occurrence of a reduced number of acceptor molecules in the enzymatic fraction.

Glucosyl donor and glucosyl acceptor specificity

Table 1 shows that UDP-Glc is the specific sugar donor for UDP-Glc: protein transglucosylase I, as previously described [l, 21. Formation of reaction I product has already been shown for the particulate [l, 21 and solubilized [lo] preparations to be unimpaired by the addition of glycogen to the incubation mixture, though incorporation of glucose into the polysaccharide was previously found [l, lo]. On the other hand, as shown in Table 1, it was found that the purified enzyme in peak I, besides its high specificity for the sugar donor UDP-Glc, is also specific for the endogenous acceptor protein, since it was not able to transfer ['4C]glucose from labeled UDP-Glc to added polysaccharide (Table 1). Thus, exogenous polysaccharide does not compete with the endogenous acceptor protein. Moreover, the lack of methanol- precipitable radioactivity when the incubation was carried out in the presence of glycogen shows the absence of starch synthetase activity in peak I (Table 1). Even increased concentrations of the glucosyl donors failed to reveal either starch synthetase activity or formation of reaction 2 product.

Characterization of the enzyme-catalyzing reaction I Since peak I contained UDP-Glc : protein transgluco-

sylase I activity as well as the endogenous acceptor protein,

Table 1. Glucosyl donor and glucosyl acceptor specificity of UDP- G1c:protein transglucosylase I The final concentration of the sugar nucleotides was 4 pM, and that of glucose-I-P was 6 pM. Each assay mixture contained 18 pg protein and incubations were for 60min. The radioactive material pre- cipitated with 10% CI3AcOH or with methanol after precipitation with C13AcOH was measured as in [I]. Glycogen (200 pg) was added as the exogenous acceptor in expt B

Expt Glucosyl donor Radioactivity incorporation

C1 SACOH - methanol- ins o 1 u b 1 e insoluble, after

precipitation C13AcOH

A. With endogenous acceptor

ADP-['4C]Gl~ 50 30 ['4C]Glucose-l-P 65 30

B. With exogenous acceptor UDP-[14C]Glc 3060 40 ADP-['4C]Glc 55 30 ['4C]Glucose-l-P 60 40

UDP-[14C]Glc 2800 35

it was of interest to determine whether the two functional components of the system, i.e. the acceptor capability and the transglucosylase activity, are located on the same molecule or whether there are two different molecules. Therefore, peak I was chromatographed on a ConA-Sepharose 4B column (Fig. 3). The elution profile obtained after affinity chromatography showed that the activity of the enzyme catalyzing reaction 1 was found in the unbound fractions (Fig. 3 A), indicating that neither the acceptor capability nor the transglucosylase activity have affinity for the immobilized lectin. A major HI04/Schiffs reagent positive band, also present in peak I (Fig. 3 B, lane l), was found by denaturing gel electrophoresis in the fractions bound to the lectin (Fig. 3 B, lane 2), which cannot be related to the enzymatic activity. Therefore, both the endogenous acceptor as well as the enzyme lack carbohydrates (glucose or mannose) or, if they are present, their binding to the lectin is prevented by their steric conformation (Fig. 3 A and Fig. 3 B, lane 3).

543

10

N

k

Fi x 5 -

V

- I I I - A

$. -

0 - 4

v 1 I

Fraction number

Fig. 3. Affinity chromatography of UDP-G1c:protein transglucosylase I on a ConA-Sepharose 4B column. (A) Peak I (1 mg) in buffer B was applied to a ConA-Sepharose 4B column, which had been equilibrated with the same buffer. The column was first washed with the equilibrium buffer and then eluted with 0.2 M methyl a-glucoside in the same buffer as indicated by the arrow in the figure, and fractions of 1 ml were collected. Enzyme activity was determined by using 150-pl aliquots of each fraction (0). (B) Urea/SDS-gel electrophoresis analysis of bound and unbound fractions from the Cod-Sepharose 4B column. Original sample of peak I (lane l), pooled bound fractions (lane 2) and pooled unbound fractions (lane 3) were subjected to urea/ SDS-gel electrophoresis (10%). About 80 pg of the protein sample was applied in each well. A photograph of the Coomassie-blue-stained gels is shown. The arrow indicates the HIO,/Schiffs reagent positive band in lanes 1 and 2

Peak I was also analyzed by gel filtration and velocity sedimentation. Gel filtration on Sephacryl S-300 shows that the activity of UDP-Glc: protein transglucosylase I elutes as a single peak with a partition coefficient of elution (Kay) of 0.24. The plot of Stokes radius against Kay [20] obtained with marker proteins gives rise to a Stokes radius of 6.1 nm (not shown). The szo,w determined for UDP-G1c:protein transglucosylase I was 10.3 S (Fig.4), while that of the native reaction 1 product, also submitted to sucrose density gradient centrifugation under the same conditions, was 8.8 S (Fig. 4). A molecular mass M, of 300 kDa was calculated for the native enzyme by combining the Stokes radius with the sedimentation coefficient (both experimentally determined) and partial specific volume data available in the literature [20].

DEAE-cellulose chromatography (Fig. l), affinity chromatography (Fig. 3), sucrose density gradient centrifugation (Fig. 4) and gel filtration indicate that the enzyme catalyzing reaction 1 failed to be separated from the endogenous acceptor. Possible explanations are that the acceptor and the

H

Fraction number Fig.4. Determination of the sedimentation coefficients of UDP- G1c:protein transglucosylase I and of reaction I product. Peak I (200 pg protein) or the incubation mixture for reaction 1 product formation after incubation for 30 min (200 pg protein) were layered on opposing sucrose density gradients and ultracentrifuged as described under Materials and Methods. Each fraction from the first gradient was assayed for enzymatic activity (0). "C-labeled reaction 1 product in the other gradient was detected by scintillation counting after precipitation of each fraction with 10% C13AcOH (0). Cytochrome c (1, szo,w = 1.7 S), alkaline phosphatase (2, s20.w = 6.3 S), lactate dehydrogenase (3, szo,w = 7.3 S) and catalase (4, szo,w = 11.3 S) were used as standards. Fractions are numbered beginning with the bottom of the gradient

enzyme are two different proteins that co-purified after each of the purification steps tested herein or that they are a single protein with enzymatic activity and acceptor capacity which is autoglucosylated. Even the lower value obtained for the sedimentation coefficient of reaction 1 product cannot discard the latter possibility (i.e. a single protein), since a conformational change may occur induced by glucosylation of the enzyme itself.

Nature of the product synthesized by UDP-G1c:protein transglucosylase I (reaction 1 product)

Reaction 1 product, obtained as described under Materials and Methods, was insensitive to ci- and B-amylase treatments and sensitive to the action of glucoamylase. As a result of this latter treatment, only radiolabeled glucose was found on paper chromatography in butanol/pyridine/water (6 : 4 : 3, by vol.). The labeled product synthesized by UDP-Glc : protein transglucosylase I is also sensitive to proteolytic digestion. To analyze this protease-sensitive product, a large-scale incubation (10-fold) was utilized to generate sufficient glucosylated product. Over 95% of the radioactivity applied to a Sephadex G-15 column was recovered in the included fractions when the products of protease digestion of labeled reaction 1 product were fractioned by exclusion chromatography. As Sephadex G-15 excluded substances with molecular masses over 1500 Da, a molecular mass below this value can be ascribed to the labeled components included. To verify that

544

Fig. 5 . Size determination of the I4C-labeled glucosylated peptide of reaction 1 product. The labeled product (100 pg protein; 2000 cpm) was submitted to urea/SDS-gel electrophoresis. Fluorograms of the labeledpeptideona 10% (A)ora12.5% (B)acrylamidegelareshown. Thc corresponding positions of protein molecular mass standards (in kDa) are indicated on each side

the included radioactive peak was [ 14C]glucose bound to peptide material, the corresponding fractions from the Sephadex G-15 column were pooled, concentrated and analyzed by paper electrophoresis in 5% formic acid at pH 2.5 and in 0.25 M sodium carbonate/sodium bicarbonate, pH 9.2. The radioactive compound obtained after proteolysis of reaction 1 product corresponded to a glucopeptide since it moved to the negative pole in acidic medium and to the positive pole in alkaline buffer.

On the other hand, a single labeled band was detected when reaction 1 product was analyzed by urea/SDS-PAGE (Fig. 5). Denaturing polyacrylamide gel electrophoresis and fluorography showed that all the radioactivity migrated as a 38-kDa polypeptide at 10% (Fig. 5A) and 12.5% acrylamide (Fig. 5 B). This apparent molecular mass does not vary at the different acrylamide concentrations used. It seems likely that this 38-kDa polypeptide is the subunit of the endogenous acceptor that becomes glucosylated.

Alkaline treatment under reducing conditions converts the peptide-linked [ ''C]glucose to labeled sorbitol as judged by paper electrophoresis and therefore serves to identify the sugar of the linkage region. This result also indicates that the enzyme catalyzing reaction 1 transfers only one glucosyl moiety to the specific aminoacyl residue of the endogenous acceptor protein, since the alkaline treatment in the presence of NaBH4 releases 0-glycosidic carbohydrate units as reduced oligosac- charides. In addition, the susceptibility to mild alkaline hy- drolysis is consistent with the occurrence of a Ser/Thr-Glc linkage.

Identijkation of the sugar-linked amino acid in reaction I product

Confirmation of the amino acids involved in the carbohydrate protein linkage may be done by reducing the P-elimination products with a tritiated reducing agent. This was done on the glucopeptide fraction obtained after proteolysis of reaction 1 product as follows. 14C-labeled reaction 1 prod-

uct (90000 cpm) was incubated with subtilisin in 0.1 M Tris/ HCl buffer, pH 8.0, 0.01 M CaClz and 0.1 M KCl. The pH was maintained with NaOH and after 24 h a second addition of protease was made and the digestion continued for 48 h. The reaction was stopped with 1 ml of 10% C1,AcOH and centrifuged. The supernatant was freed from the acid by ether extraction and subjected to exclusion chromatography on a Sephadex G-15 column (40 x 0.5 cm). The included radioactive fractions were pooled and submitted to paper electrophoresis in 5% formic acid, pH 2.5. The radioactive area (glucopeptide material) was eluted from the paper and treated with 7mM NaB3H4 (884 Ci/mol, Amersham In- ternational, England) in 0.5 M NaOH and 3 mM PdClz as catalyst [21] at 50°C for 5 h. The reaction mixture was then carefully acidified with acetic acid in order to decompose the excess tritiated borohydride. The mixture was dried under reduced pressure and the excess boric acid was removed as methyl borate. Then it was brought to dryness and redissolved in water. The procedure was repeated several times in order to remove any exchangeable 3H. The mixture was passed through a small column of Dowex 50W-X8 (H+ form) and then an aliquot (l/lO) was submitted to paper electrophoresis in 5% formic acid, pH 2.5. This treatment produced three main peaks of radioactivity; two of these coincided with alanine and 2-aminobutyric acid, which are expected to be formed by the NaB3H4 reduction of the 2-aminoacrylic acid and 2-aminocrotonic acid, which would be generated by p-elimination of carbohydrates attached to serine and threonine residues by 0-glycosidic bonds. The sharp peak of radioactivity near the origin migrates far too slowly to be free amino acids and no attempt was made to characterize it. It seems probable that the glucopeptide fraction that was submitted to the NaB3H4 treatment also contained, in addition to glucose linked to serine or threonine, oligopeptides of variable length, which contained glucose linked to serine or threonine. Thus, as a result of this alkaline reductive treatment, together with tritiated alanine or 2-aminobutyric acid, short-chain tritiated oligopeptides may also be obtained. In addition, by-products from alkaline degradation reactions of glucose have to be considered as well, since the above treatment was performed in the presence of very low concentrations of tritiated sodium borohydride (of high specific radioactivity) to assure the labeling of the minute amounts of amino acids involved in the 0-glucosidic linkage. In addition, the f l- elimination reaction, and in particular the conversion of serine and threonine into alanine and 2-aminobutyric acid, respectively, are rarely quantitative, as has been reported elsewhere

The areas corresponding to alanine and 2-aminobutyric acid were eluted from the paper and the identity of these amino acids was confirmed by one- and two-dimensional TLC on silica gel (Fig. 6). However, there were insufficient amounts of material recovered from the plate to characterize 2-aminobutyric acid further by two-dimensional TLC.

The concentration of the reducing agent was maintained at an unusually low level with the aim of identifying the reduced amino acids, even through preservation of the linking sugar does not occur. This was done because of the low quanti- ties of glucopeptide material available.

Recently, Aon and Curtino [23] and Rodriguez and Whelan [24] have reported that the hydroxyl group of tyrosine is involved in the attachment of glycogen to protein. It is noteworthy that the codons for Tyr and Ser differ by a single base substitution. Sharon [22] has pointed out that the codons for the amino acids (Asn, Ser and Thr) to which carbohydrates

P21.

545

Fig. 6. Two-dimensional TLC of the tritiated substances following alkaline tritiated horohydride treatment. The radioactive peak eluted from the one-dimensional TLC was separated by two-dimensional TLC in silica gel (1st dimension, from bottom to top) in n-butanoll acetone/ammonia/water (10: 105: 2) and in isopropanol/acetic acid/ water (20: 1 : 5) (2nd dimension, from left to right). The fluorogram of the silica gel plate is shown. The dashed circles indicate the positions of the standard amino acids (Abu = 2-aminobutyric acid), as visualized with an ultraviolet light after the fluorescamine stain

are linked in proteins differ among themselves by only single base substitutions.

Coupled with the extensive characterization of the isolated enzyme (Table 1 and Fig. 3), this work represents a significant advance in our knowledge of the first step in protein-bound a-glucan synthesis in the potato tuber. Further experimental data on the primer ability of the reaction 1 product to be used by specific starch synthetases and phosphorylases (reaction 2) are the subject of a forthcoming report.

The authors are indebted to the members of the Instituto de Investigaciones Bioquimicas ‘Fundacion Campomar ’ for helpful dis-

cussion and criticisms. J. S. T. is a Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina. The data in this report have been submitted as partial fulfillment of the requirement for the Ph. D. degree by S. M.

REFERENCES 1. 2.

3.

4.

5.

6.

7.

8.

9.

10. 11.

12.

13.

14.

35. 16.

17.

18.

19.

20.

21. 22.

23. 24.

Lavintman, N. & Cardini, C. E. (1973) FEBS Lett. 29,43-46. Lavintman, N., Tandecan, J., Carceller, M., Mendiara, S. &

Krisman, C. R. & Barengo, R. (1975) Eur. J. Biochem. 52, 117-

Blumenfeld, M. L., Whelan, W. J. & Krisman, C. R. 1983) Eur.

Aon, M. A. & Curtino, J. A. (1984) Eur. J. Biochem. 140, 557-

Barengo, R., Flawia, M. M. & Krisman, C. R. (1975) FEBS Lett.

Tomos, A. D. & Northcote, D. H. (1978) Biochem. J. 174,283-

Tandecarz, J. S. & Cardini, C. E. (1978) Biochim. Biophys. Acta

Tandecarz, J. S . & Cardini, C. E. (1979) Pfant Sci. Lett. 15,151 -

Moreno, S. & Tandecarz, J. S. (1982) FEBS Lett. 139, 313-316. Thomas, J. A., Kaith, K., Schlender, K. & Larner, J. (1968) Anal.

Tandecarz, J. S . , Lavintman, N. & Cardini, C. E. (1975) Biochim.

Tandecarz, J . S . , Sivak, M. N. & Cardini, C. E. (1978) Biochem.

Ziegler, A,, Harrison, S. C. & Liberman, R. (1974) Virology 59,

Chamberlain, J. P. (1979) Anal. Biochem. 98,132-135. Trevelyan, W. E., Procter, D. F. & Harrison, J. S. (1950) Nature

(Lond.) 166,444-445. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber,

W. & Weigele, M. (1972) Science (Wash. DC) 178,871 -872. Lowry, 0. H., Rosebrough, J. J., Farr, A. L. & Randall, R. J.

(1951) J . Biol. Chem. 193, 265-275. Roden, L. & Schwartz, N. B. (1975) in Biochemistry of

carbohydrates (Whelan, W. J., ed.) vol.5, pp.95-152, Butterworths, London and University Park Press, Baltimore.

Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta 112,

Tanaka, K. & Pigman, W. (1965) J. Biol. Chem. 240,1487- 1488. Sharon, N. (1975) in Complex Carbohydrates. Their chemistry,

biosynthesis and functions, pp. 65 - 83, Addison Wesley Publishing Co., Reading MA.

Cardini, C. E. (1974) Eur. J . Biochem. 50, 145-155.

123.

J . Biochem. 135, 175-179.

566.

53,274-278.

290.

S43,423 - 429.

158.

Biochem. 25,486-499.

Biophys. Acta 399, 345-355.

Biophys. Res. Commun. 82,157 - 164.

509 - 51 5.

346 - 362.

Aon, M. A. & Curtino, J. A. (1985) Biochem. J. 229,269-272. Rodriguez, I. R. & Whelan, W. J. (1985) Biochem. Biophys. Res.

Commun. 132,829 - 836.

α-glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglucosylase i :...

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